Identification of functional bradykinin B(2) receptors endogenously expressed in HEK293 cells.
The human embryonic kidney (HEK) 293 cell line is widely used in cell biology research. Although HEK293 cells have been meticulously studied, our knowledge about endogenous G protein-coupled receptors (GPCR) in these cells is incomplete. While studying the effects of bradykinin (BK), a potent growth factor for renal cells, we unexpectedly discovered that BK activates extracellular signal-regulated protein kinase 1 and 2 (ERK) in HEK293 cells. Thus, we hypothesized that HEK293 cells possess endogenous BK receptors. RT-PCR demonstrated the presence of mRNAs for BK B(1) and BK B(2) receptors in HEK293 cells. Western blotting with BK B(1) and BK B(2) receptor antibodies confirmed this result at the protein level. To establish that BK receptors are functional, we employed fluorescent measurements of intracellular Ca(2+), measured changes in extracellular acidification rate (ECAR) as a reflection of the Na(+)/H(+) exchange (NHE) with a Cytosensortrade microphysiometer, and assessed ERK activation by Western blotting with a phospho-specific ERK antibody. Exposure of HEK293 cells to BK produced a concentration-dependent rise in intracellular Ca(2+) (EC(50)=36.5+/-8.0 x 10(-9)M), a rapid increase in tyrosine phosphorylation of ERK (EC(50)=9.8+/-0.4 x 10(-9)M), and elevation in ECAR by approximately 20%. All of these signals were blocked by HOE-140 (B(2) receptor antagonist) but not by des-Arg(10)-HOE-140 (B(1) receptor antagonist). We conclude that HEK293 cells express endogenous functional BK B(2) receptors, which couple to the mobilization of intracellular Ca(2+), increases in ECAR and increases in ERK phosphorylation.
Kramarenko
Inga I
II
Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC 29401, USA.
Bunni
Marlene A
MA
Morinelli
Thomas A
TA
Raymond
John R
JR
Garnovskaya
Maria N
MN
D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002460:Cell Line; D017931:DNA Primers; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney / Q000196:embryology; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D043783:Receptor, Bradykinin B1 / Q000235:genetics / Q000502:physiology; D043782:Receptor, Bradykinin B2 / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D043783:Receptor, Bradykinin B1; D043782:Receptor, Bradykinin B2; D012313:RNA; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium